The human rod opsin gene (RHO) harbors many mutations that cause retinitis pigmentosa (RP). The folded opsin mRNA is a target for mutation-independent hammerhead ribozyme (hhRz) gene therapy for autosomal dominant (ad) RP. We have achieved a single highly potent hhRz candidate therapeutic for adRP due to all known human RHO mutations. The long-range goal is to translate this effective hhRz therapeutic for RHO adRP into human clinical trials. The objective is to conduct a preclinical proof-of-materials test of this hhRz agent in mouse models that have retinal degeneration on the basis of expression of human mutant and wild type (WT) RHO genes to model human adRP in the absence of mouse RHO gene expression. The central hypothesis is that the reduction of toxic mutant RHO mRNA and protein will reduce photoreceptor stresses and reduce the rate of retinal degeneration due to mutant RHO allele expression. The rationale is that reduction of the mutant (P347S) RHO mRNA and protein by the mutation-independent hhRz agent must be combined with approaches and strategies to reconstitute WT RHO expression (also reduced by the hhRz) to avoid haploinsufficiency that would also compromise photoreceptor vitality. To test the central hypothesis and accomplish the objective three Specific Aims will be pursued: Aim 1. Conduct a simple preclinical test of the Knockdown hhRz strategy by testing for rescue of retinal degeneration in a partially humanized murine model of adRP that expresses a human mutant rod opsin transgene on the mouse WT RHO background. Expected results are that suppression of toxic mutant RHO mRNA/protein will rescue photoreceptors and ameliorate retinal degeneration. Aim 2. Test the hhRz PTGS agent for toxicity in a fully humanized mouse model that expresses only normal human RHO in mouse rod photoreceptors. Expected results are knowledge of the minimum amount of WT human rhodopsin that is needed to keep a mammalian rod photoreceptor alive and vital. Aim 3. Test the lead hhRz agent and a reconstituting human WT RHO allele for rescue in a humanized adRP model in which human mutant and WT RHO mRNAs are expressed in mouse rod photoreceptors. Expected results in the fully humanized mouse model of adRP are rescue from retinal degeneration without sustained therapeutic haploinsufficiency. The proposed plan is innovative in that it uses mouse models developed in this lab that are humanized for target mRNA to simulate human clinical trial, it uses aptamer technology to modulate activity of the therapeutic hhRz using to optimize knockdown vs. toxicity in vivo, and it uses state-of-the-art vector delivery approaches. The significance is that successful rescue in humanized mouse models of adRP would provide safety and efficacy outcomes to support clinical translation of this gene therapy approach for human RHO adRP. Our technology platform allows us to rapidly develop potent hhRz agents to any known disease target mRNA in which knockdown is expected to ameliorate the disease process. PUBLIC HEALTH RELEVANCE: The proposed experimental plan is significant and relevant to a mission of the National Eye Institute to develop treatments for hereditary retinal degenerations. Here we conduct a preclinical test of highly potent post-transcriptional gene silencing agents (e.g. ribozymes) developed in this lab to rescue retinal degeneration in humanized mouse models with rod opsin mutations. A success to rescue retinal degeneration in a unique fully humanized mouse model of autosomal dominant retinitis pigmentosa could potentially support translation of successful therapeutic agents into human clinical trials.